Cell surface receptors Ly-9 and CD84 recruit the X-linked lymphoproliferative disease gene product SAP by Joan Sayós, Margarita Martı́n, Alice Chen, Marı́a Simarro, Duncan Howie, Massimo Morra, Pablo Engel, and Cox Terhorst Blood Volume 97(12):3867-3874 June 15, 2001 ©2001 by American Society of Hematology

The cell surface molecule 2B4 recruits the XLP gene product SAP after phosphorylation of the tyrosine residues in its cytoplasmic tail. The cell surface molecule 2B4 recruits the XLP gene product SAP after phosphorylation of the tyrosine residues in its cytoplasmic tail. (A) Comparison of the interactions between 2B4/SAP and SLAM/SAP in yeast cells. To detect binding between the cytoplasmic tail of 2B4 and SAP, a yeast 2-hybrid system was adapted to measure interactions with phospho-proteins. To this end, mutations of c-fyn were cotransfected with SAP into the yeast cell. Binding between 2 proteins in yeast cell extracts was detected by a β-galactosidase assay, as described in “Materials and methods.” For each construct, at least 3 independent colonies were tested in the β-galactosidase assay. Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of 2B4 or SLAM; solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of 2B4 or SLAM; hatched bars: cells transfected with pBRIDGE-SAP and Fyn420, 531 Y-F and with pGAD424 encoding the cytoplasmic tail of 2B4 or SLAM; dotted bars: cells transfected with pBRIDGE-SAP and Fyn 420, 531 Y-F, 176 R-Q and with pGAD424 encoding the cytoplasmic tail of 2B4 or SLAM. Control, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (B) SAP binds to phosphorylated 2B4 in NK cell line. Interactions between SAP and 2B4 in the YT cell line were studied by immunoprecipitation of 2B4 followed by Western blot analysis with anti-SAP. YT cells (50 × 106 cells/mL) were biotinylated and then incubated in the presence or absence of 1 mM pervanadate. Cells were lysed in detergent, and 2B4 was immunoprecipitated with the 2B4 specific monoclonal antibody C1.7 (α-2B4). After SDS-PAGE, proteins were transferred to a PVDF membrane and were identified with streptavidin, antiphosphotyrosine (α-PY), or antihuman SAP 10C4.2 (α-SAP). CONTROL, immunoprecipitation with an irrelevant monoclonal antibody. (C) SAP binds to nonphosphorylated and phosphorylated SLAM in a T-cell line. Interactions between SAP and SLAM were studied in the T-cell transfectant cell line EL-4/SLAM4 by immunoprecipitation of SLAM, followed by Western blot analysis with anti-SAP. EL-4/SLAM4 cells (20 × 106 cells/mL) expressing human SLAM were treated with pervanadate, as described in panel B, and SLAM was immunoprecipitated by an antihuman SLAM monoclonal antibody (α-SLAM).2 After SDS-PAGE, proteins were transferred to a PVDF membrane and identified by Western blot analysis with a rabbit antihuman SLAM antibody, antiphosphotyrosine (α-PY), or antihuman SAP (α-SAP) 10C4.2 CONTROL, immunoprecipitation with an irrelevant monoclonal antibody. Joan Sayós et al. Blood 2001;97:3867-3874 ©2001 by American Society of Hematology

SAP interacts with the phosphorylated cytoplasmic tail of Ly-9 SAP interacts with the phosphorylated cytoplasmic tail of Ly-9.A human cDNA library made from poly A+ RNA of the human T-cell line KT3 in pGAD424 was screened with the altered yeast 2-hybrid system. SAP interacts with the phosphorylated cytoplasmic tail of Ly-9.A human cDNA library made from poly A+ RNA of the human T-cell line KT3 in pGAD424 was screened with the altered yeast 2-hybrid system. Thus, 5 cDNA clones encoding the cytoplasmic tail of Ly-9 were isolated; an example is shown in panel A. To map the binding sites in the cytoplasmic tail of human Ly-9, 3 mutations of Ly-9 were analyzed in panel B. SAP binding to phospho-Ly-9 was shown in murine thymocytes in panel C. (A) Interactions of SAP and the cytoplasmic tail of Ly-9 in yeast are dependent on the presence of Fyn 420, 531 Y-F. The interaction of SAP with the cytoplasmic tail of Ly-9 in the presence or absence of Fyn 420, 531 Y-F took place in the yeast cell and was measured in a β-galactosidase assay. For each construct, at least 3 independent colonies were tested in the galactosidase assay. Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of Ly-9; solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of Ly-9; hatched bars: cells transfected with pBRIDGE-SAP and Fyn420, 531 Y-F and with pGAD424 encoding the cytoplasmic tail of Ly-9. Control, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (B) SAP interacts with 2 phosphotyrosine motifs in the cytoplasmic tail of Ly-9. The interaction of SAP with 3 Ly-9 cytoplasmic tail mutations in the presence or absence of Fyn420, 531 Y-F took place in the yeast cell and was measured in a β-galactosidase assay. For each construct, at least 3 independent colonies were tested in the galactosidase assay. The cytoplasmic tail of Ly-9 was mutated in tyrosine residue 558 (558-YF), tyrosine 581 (581-YF), or in both tyrosine 558 and tyrosine 581 (558-581-YF). Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of Ly-9. Solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of Ly-9 or one of the Ly-9 mutants. Hatched bars: cells transfected with pBRIDGE-SAP and Fyn420, 531 Y-F and with pGAD424 encoding the cytoplasmic tail of Ly-9 or one of the Ly-9 mutants. CONTROL, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (C) Association of SAP and Ly-9 in mouse thymocytes. Mouse thymocytes (50 × 106 cells/mL) were biotinylated and then incubated in the presence or absence of 1 mM pervanadate. Cells were lysed, and Ly-9 was immunoprecipitated with 1 μg antimouse Ly-9 monoclonal antibody (IP aLy9). Proteins were transferred to PVDF, and Western blot analysis (WB) identified specific proteins with streptavidin, antiphosphotyrosine (WB α-PY) and a rabbit antimouse SAP antibody (WB α-SAP). CONTROL, immunoprecipitation with a monoclonal hamster antibody. Joan Sayós et al. Blood 2001;97:3867-3874 ©2001 by American Society of Hematology

Interactions of SAP with the phosphorylated cytoplasmic tail of CD84 Interactions of SAP with the phosphorylated cytoplasmic tail of CD84.(A) CD84 and SAP in yeast. Interactions of SAP with the phosphorylated cytoplasmic tail of CD84.(A) CD84 and SAP in yeast. The interaction of SAP with the cytoplasmic tail of human CD84 in the presence or absence of Fyn 420, 531 Y-F took place in the yeast cell and was measured in a β-galactosidase assay. For each construct, at least 3 independent colonies were tested in the galactosidase assay. Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of CD84; solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of CD84; hatched bars: cells transfected with pBRIDGE-SAP and Fyn420, 531 Y-F and with pGAD424 encoding the cytoplasmic tail of CD84. Control, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (B) SAP coprecipitates with phosphorylated CD84 in a B-cell line. Cells of a variant of the Burkitt lymphoma cell line Raji, which was known to express SAP in its cytoplasm,2 were biotinylated and incubated in the presence or absence of 1 mM pervanadate. After lysis in detergent, CD84 was immunoprecipitated with 1 μg antihuman CD84 monoclonal antibody (IP α-CD84) (CD84.1.21). Proteins were transferred to PVDF membranes and Western blotted (WB) with streptavidin (WB streptavidin), antiphosphotyrosine (α-pY), and monoclonal antihuman SAP (α-SAP)10C4.2 CONTROL, immunoprecipitation with mouse immunoglobulin. (C) SAP coprecipitates with phosphorylated CD84 in Jurkat. Stable CD84 transfectants of the T- cell line Jurkat were biotinylated and then incubated in the presence or absence of 1 mM pervanadate. After lysis in detergent, CD84 was immunoprecipitated with 1 μg antihuman CD84 monoclonal antibody (IP α-CD84) (CD84.1.21). Proteins were transferred to PVDF membranes and Western blotted (WB) with streptavidin (WB streptavidin), antiphosphotyrosine (α-pY) and monoclonal antihuman SAP (α-SAP) (10C4.2). CONTROL, immunoprecipitation with mouse immunoglobulin. Joan Sayós et al. Blood 2001;97:3867-3874 ©2001 by American Society of Hematology

Schematic representation of the cytoplasmic domains of 4 SLAM family members.SAP has been shown to bind preferentially to a consensus binding motif: T (I/V)Y x x (V/I), where x represents any amino acid. Schematic representation of the cytoplasmic domains of 4 SLAM family members.SAP has been shown to bind preferentially to a consensus binding motif: T (I/V)Y x x (V/I), where x represents any amino acid. The cytoplasmic domains of the SLAM family members SLAM, 2B4, Ly9, and CD84 contain multiple SAP-binding motifs. Lines indicate the cytoplasmic domain of the SLAM family members. Blocks represent real (*) or putative SAP-binding motifs. Blocks labeled with # are putative motifs proven to be unable to bind SAP, whereas those without marks have not been tested for SAP binding. Joan Sayós et al. Blood 2001;97:3867-3874 ©2001 by American Society of Hematology

SAP blocks binding of SHP-2 to phosphorylated Ly-9 and CD84 SAP blocks binding of SHP-2 to phosphorylated Ly-9 and CD84.To detect interactions between Ly-9 or CD84 and SHP-2, COS-7 cells were cotransfected with cDNAs encoding one of the receptors, humanc-fyn, in the presence or absence of human SAP. On immunoprecipi... SAP blocks binding of SHP-2 to phosphorylated Ly-9 and CD84.To detect interactions between Ly-9 or CD84 and SHP-2, COS-7 cells were cotransfected with cDNAs encoding one of the receptors, humanc-fyn, in the presence or absence of human SAP. On immunoprecipitation with monoclonal antibody directed at either Ly-9 or CD84, the receptor-associated protein complexes were analyzed by Western blotting. (A) SHP-2 binds to phosphorylated Ly-9 in the absence of SAP. COS-7 cells were transfected with the indicated constructs, and 48 hours later cell surface proteins were biotinylated, cells were lysed, and Ly-9 was immunoprecipitated as described in “Materials and methods.” Immunoprecipitates and whole-cell lysates were run on SDS-PAGE and transferred to PVDF. Ly-9 was detected using streptavidin conjugated to horseradish peroxidase, and phosphotyrosine, SAP, and SHP-2 were detected using the antibodies described in “Materials and methods.” Whole-cell lysates were Western blotted for SAP and SHP-2 to control for gel loading. (B) SHP-2 binds to phosphorylated CD84 in the absence of SAP. COS-7 cells were transfected with the indicated constructs, and 48 hours later cell surface proteins were biotinylated, cells were lysed, and CD84 immunoprecipitated as described in “Materials and methods.” Immunoprecipitates and whole-cell lysates were run on SDS-PAGE and transferred to PVDF. CD84 was detected using streptavidin conjugated to horseradish peroxidase, and phosphotyrosine, SAP, and SHP-2 were detected using the antibodies described in “Materials and methods.” Whole-cell lysates were Western blotted for SAP and SHP-2 to control for gel loading. Joan Sayós et al. Blood 2001;97:3867-3874 ©2001 by American Society of Hematology

Phosphorylated CD84 and Ly-9 associate with SAP at the plasma membrane Phosphorylated CD84 and Ly-9 associate with SAP at the plasma membrane.To verify the results obtained with the yeast 2-hybrid system and immunoprecipitation in COS-7 cells, a fluorescence microscopy approach was used. Phosphorylated CD84 and Ly-9 associate with SAP at the plasma membrane.To verify the results obtained with the yeast 2-hybrid system and immunoprecipitation in COS-7 cells, a fluorescence microscopy approach was used. COS-7 cells were transfected with SAP and Fyn along with CD84 or Ly-9. The cells were then stained for SAP and Ly-9 or CD84. Colocalization of SAP and Ly-9 or CD84 at the cell membrane can be visualized by yellow fluorescence where the 2 fluorochromes colocalize. Joan Sayós et al. Blood 2001;97:3867-3874 ©2001 by American Society of Hematology

CD84 and Ly-9 colocalize with SAP in Jurkat after activation of the receptors with monoclonal antibody.A fluorescence microscopy approach was used in the human T-cell line Jurkat to confirm the SAP/Ly-9 and SAP/CD84 interaction in T cells. CD84 and Ly-9 colocalize with SAP in Jurkat after activation of the receptors with monoclonal antibody.A fluorescence microscopy approach was used in the human T-cell line Jurkat to confirm the SAP/Ly-9 and SAP/CD84 interaction in T cells. Jurkat cells were incubated with 5 μg/mL biotinylated anti-CD84 or anti-Ly9 antibody for 30 minutes at 4°C, as detailed in “Materials and methods.” Cells were then incubated with streptavidin-FITC and incubated at 4°C (control) or 10 minutes at 37°C (capping). Cells were then stained with Cy3-SAP, as indicated. Green fluorescence shows CD84 (A, C) or Ly 9 (E, G). Staining with red fluorescence indicates SAP (B, D, F, H). White arrows show colocalization. Joan Sayós et al. Blood 2001;97:3867-3874 ©2001 by American Society of Hematology

Summary of the SAP-binding proteins Summary of the SAP-binding proteins.Binding of SAP to the cytoplasmic tail of the cell surface receptors is indicated by a plus sign, and absence of binding to SAP is indicated by a minus sign. Summary of the SAP-binding proteins.Binding of SAP to the cytoplasmic tail of the cell surface receptors is indicated by a plus sign, and absence of binding to SAP is indicated by a minus sign. SAP binds to the phosphorylated (pY) cytoplasmic tails of SLAM, 2B4, Ly-9, and CD84 and only to the nonphosphorylated (Y) tail of SLAM. CD48 is the ligand for 2B4 (see “Discussion”). B, B lymphocyte; T, T lymphocyte; CD8, CD8+ lymphocyte; COOH, carboxy-terminus; DC, dendritic cell; Mφ, macrophage; NH2, amino-terminus; M, SAP-binding motif; –, N-linked carbohydrate side chain. Joan Sayós et al. Blood 2001;97:3867-3874 ©2001 by American Society of Hematology